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| | Fluorescence-Based APE1 Endonuclease Activity Assay |
| Description: | In this assay, the complementary single-stranded oligonucleotides at 100 m were mixed at a 1:1 ratio and annealed in assay buffer (50 mm Tris pH 7.5, 25 mm NaCl, 2 mm MgCl2, 1 mm DTT, 0.01% Tween-20) at 95 °C for 5 min to generatethe double-stranded substrate of APE1.[A. Simeonov, A. Kulkarni, D. Dorjsuren, A. Jadhav, M. Shen, D. R. McNeill, C. P. Austin, D. M. Wilson 3rd, PLoS ONE 2009, 4, p. e5740.] After the annealing, the reaction mixture was allowed to cool to room temperature. The assay was carried out in 96-well black plates with flat bottom (Costar, Corning Inc., NY, USA) at a finalreaction volume of 100 μL. The reaction mixtures containing 2.5 U of human recombinant APE1 (6 μL) (New England Biolabs, Ipswich, MA, USA) were incubated in the assay buffer abovementioned, in the absence or presence of the putative APE1 inhibitors (5 μL), at room temperature for 15 min. The reaction was initiated by the addition of double-stranded substrate (6 μL) to obtain the final concentration of 250 nm. DMSO was maintained at a concentration of 0.5% (v/v) in all assays (negative control). Fluorescence readings were acquired at 1-min intervals over an incubation period of 30 min at 37 °C using a SpectraMax Gemini EM microplate plate reader (Molecular Devices, Berkshire, UK) in the kinetic mode at excitation wavelength 550 nm, emission wavelength 584 nm, and cutoff at 570 nm. |
| Affinity data for this assay | |
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